Antipoxvirus drug targets are a pressing issue, given the concern that undeclared stocks of smallpox might be used as a bioterror weapon. Whereas the eradication of smallpox was a triumph of prophylactic immunization, the treatment of smallpox never advanced beyond supportive therapy. Treatments for other poxvirus infections of humans (molluscum contagiosum, monkeypox, and complications of immunization with vaccinia virus) are also either nonspecific or nonexistent. The outbreak of human monkeypox infections in the US Midwest in 2003 highlighted the risks of re,emergence of human poxvirus disease. Our goal is to identify novel drugs for the treatment and chemoprophylaxis of smallpox by blocking the transcription and capping of viral mRNAs. We will screen LifePharms' unique and proprietary library of extracts from >13,000 wild mushrooms to discover new inhibitors of poxvirus replication targeted to the mRNA transcription apparatus packaged within the core of the infectious virion. The in vitro transcription reaction of permeabilized virions is generally accepted to faithfully recapitulate the process of viral early mRNA biogenesis as it occurs in the host cell. By screening in vitro for inhibition of mRNA synthesis and processing by permeabilized virions, we expect to identify candidate antivirals that block one or more of the key viral enzymes responsible for transcription and capping of poxvirus early mRNAs. This strategy for primary screening has key advantages over screens against individual viral proteins because: (i) it selects for compounds that are capable of accessing the target within the virion core; and (ii) it embraces multiple potential enzymatic targets within a single assay platform. The targets include: (i) DNA-dependent RNA polymerase; (ii) ETF (early transcription factor), a DNA-dependent ATPase; (iii) the capping enzymes RNA triphosphatase, RNA guanylyltransferase and RNA guanine-N7 methyltransferase; (iv) NPH1, a transcription elongation/termination factor with DNA-dependent ATPase activity; (v) NPH2, an RNA helicase; and (vi) DNA topoisomerase. These poxvirus enzymes are outstanding drug targets and specific inhibitors of these enzymes will provide lead compounds for drug development as well as key tools for basic studies of poxvirus replication. Although fungal natural products has made major contributions to pharmacology and drug discovery, only a fraction of all fungal species have been screened for bioactive compounds. LifePharms' library contains species diversity nearly equivalent to all fungal species examined previously. The drug discovery project outline here merges the complementary expertise of LifePharms, Inc. in fungal ecology and natural product extract acquisition, Dr. Stewart Shuman in poxvirus enzymology and molecular biology, and Research Triangle Institute in natural product dru 9 discovery and medicinal chemistry.